Talk:Knight:Beta-galactosidase assay/96 well format
A420 versus o-nitrophenol concentration
- Make up a solution of 7 mL to control for background absorbance in β-galactosidase assays
- 800 μL permeabilization solution
- 6 mL substrate solution without ONPG
- 200 μL EZ rich media supplemented with kanamycin and AHL
- Make up 1mL of 4 mg/mL ONP.
- Add 350 μL of 4mg/mL solution to the first well.
- Move 175 μL of previous well to next well.
- Add 175 μL of background solution to that well.
- Repeat until the well's solution looks totally clear.
- Measure A420 of each well in the plate reader.
4mg/mL -> 2mg/mL -> 1mg/mL -> 0.5 mg/mL -> 0.25 mg/mL -> 0.125 mg/mL -> 62.5 μg/mL -> 31.25 μg/mL -> 15.625 μg/mL
-> 7.8125 μg/mL -> 3.90625 μg/mL -> 1.953125 μg/mL -> 0.9765625 μg/mL